Bicistronic aav vectors encoding hexosaminidase alpha and beta-subunits and uses thereof

Abstract

Aspects of the disclosure relate to bicistronic AAV nucleic acid constructs comprising a transgene encoding hexosaminidase A (HEXA) and hexosaminidase (HEXB) proteins. In some embodiments, the disclosure provides methods for treating or preventing lysosomal storage disorders, such as Tay-Sachs disease and Sandhoff disease, using bicistronic nucleic acid constructs described by the disclosure.

What is claimed is: 
     
         1 . An isolated nucleic acid construct, comprising
 (i) a first expression cassette, comprising a nucleic acid encoding a hexosaminidase alpha-subunit (HEXA) under the control of a first promoter, and   (ii) a second expression cassette, comprising a nucleic acid encoding a hexosaminidase beta-subunit (HEXB) under the control of a second promoter,   
       wherein the first expression cassette and the second expression cassette are flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). 
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein a first intron is present between the first promoter and the sequence of the nucleic acid encoding the hexosaminidase alpha-subunit (HEXA). 
     
     
         3 . The isolated nucleic acid of  claim 1 or 2 , wherein a second intron is present between the second promoter and the sequence of the nucleic acid encoding the hexosaminidase beta-subunit (HEXB). 
     
     
         4 . The isolated nucleic acid construct of any one of  claims 1 to 3 , wherein the HexA comprises an amino acid sequence as set forth in SEQ ID NO: 1. 
     
     
         5 . The isolated nucleic acid construct of any one of  claims 1 to 4 , wherein the HexB comprises an amino acid sequence as set forth in SEQ ID NO: 2. 
     
     
         6 . The isolated nucleic acid construct of any one of  claims 1 to 5 , wherein the first intron is positioned between the first promoter and the nucleic acid sequence encoding the HexA, optionally wherein the first intron is a chimeric intron. 
     
     
         7 . The isolated nucleic acid construct of any one of  claims 1 to 6 , wherein the first promoter is positioned proximal to an AAV ITR, optionally wherein the first promoter is positioned between an AAV ITR and a nucleic acid sequence encoding HexA. 
     
     
         8 . The isolated nucleic acid construct of any one of  claims 1 to 7 , wherein the first promoter and/or the second promoter is a P2 promoter. 
     
     
         9 . The isolated nucleic acid construct of any one of  claims 1 to 8 , wherein the second intron is positioned between the second promoter and the nucleic acid sequence encoding the HexB, optionally wherein the second intron is a chimeric intron. 
     
     
         10 . The isolated nucleic acid construct of any one of  claims 1 to 9 , wherein the second promoter is positioned proximal to an AAV ITR, optionally wherein the second promoter is positioned between an AAV ITR and a nucleic acid sequence encoding HexB. 
     
     
         11 . The isolated nucleic acid construct of any one of  claims 1 to 10 , wherein the first expression cassette comprises a first poly A signal operably linked to the nucleic acid sequence encoding HexA, optionally wherein the first poly A signal is a BGH poly A signal. 
     
     
         12 . The isolated nucleic acid construct of any one of  claims 1 to 11 , wherein the second expression cassette comprises a second poly A signal operably linked to the nucleic acid sequence encoding HexB, optionally wherein the second poly A tail is an SV40 poly A signal. 
     
     
         13 . The isolated nucleic acid construct of any one of  claims 1 to 12 , wherein the first poly A signal and the second poly A signal are positioned adjacent to one another. 
     
     
         14 . An isolated nucleic acid construct, comprising
 (i) a first expression cassette, comprising a nucleic acid encoding a hexosaminidase alpha-subunit, and   (ii) a second expression cassette, comprising a nucleic acid encoding a hexosaminidase beta-subunit,   
       wherein the first expression cassette and the second expression cassette are operably linked by a bidirectional promoter, and wherein the first expression cassette and the second expression cassette are flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). 
     
     
         15 . The isolated nucleic acid construct of  claim 14 , wherein the HexA comprises an amino acid sequence as set forth in SEQ ID NO: 1. 
     
     
         16 . The isolated nucleic acid construct of  claim 14 or 15 , wherein the HexB comprises an amino acid sequence as set forth in SEQ ID NO: 2. 
     
     
         17 . The isolated nucleic acid construct of any one of  claims 14 to 16 , wherein the bidirectional promoter comprises at least one chicken beta-actin (CBA) promoter. 
     
     
         18 . The isolated nucleic acid construct of  claim 17 , wherein the bidirectional promoter comprises two CBA promoters, wherein the CBA promoters initiate transcription of the first expression cassette and the second expression cassette in opposite directions. 
     
     
         19 . The isolated nucleic acid construct of  claim 18 , wherein the bidirectional promoter comprises a CMV enhancer sequence, optionally wherein the CMV enhancer sequence is positioned between the two CBA promoters. 
     
     
         20 . The isolated nucleic acid construct of any one of  claims 14 to 19 , wherein the first expression construct comprises a first poly A signal, optionally wherein the first poly A signal is proximal to an AAV ITR. 
     
     
         21 . The isolated nucleic acid construct of any one of  claims 14 to 20 , wherein the first expression construct comprises a second poly A signal, optionally wherein the second poly A signal is proximal to an AAV ITR. 
     
     
         22 . The isolated nucleic acid construct of any one of  claims 14 to 21 , wherein the first and/or second poly A signal is each selected from SV40 poly A signal, rabbit beta-globulin (RBG) poly A signal, and bovine growth hormone (BGH) poly A signal. 
     
     
         23 . An isolated nucleic acid comprising the sequence set forth in any one of SEQ ID NOs: 3-9. 
     
     
         24 . A recombinant AAV (rAAV) comprising:
 (i) a capsid protein; and   (ii) an isolated nucleic acid of any one of claims  1  to  23 .   
     
     
         25 . The rAAV of  claim 24 , wherein the capsid protein is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, and AAV.PHP.B. 
     
     
         26 . The rAAV of  claim 24 or 25 , wherein the isolated nucleic acid comprises an ITR selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, or AAV6 ITR. 
     
     
         27 . A host cell comprising the isolated nucleic acid of any one of  claims 1 to 23 , or the rAAV of any one of  claims 22 to 24 . 
     
     
         28 . The host cell of  claim 27 , wherein the host cell is a mammalian cell, yeast cell, bacterial cell, insect cell, plant cell, or fungal cell. 
     
     
         29 . A method for treating a lysosomal storage disease, the method comprising administering the isolated nucleic acid of any one of  claims 1 to 23 , or the rAAV of any one of  claims 24 to 26 , to a subject having or suspected of having a lysosomal storage disease. 
     
     
         30 . The method of  claim 29 , wherein the lysosomal storage disease is Tay-Sachs disease or Sandhoff disease. 
     
     
         31 . The method of  claim 29 or 30 , wherein the subject is characterized as having a mutation in a HEXA gene resulting in reduced or loss of function of a hexosaminidase alpha-subunit of the subject. 
     
     
         32 . The method of  claim 29 or 30 , wherein the subject is characterized as having a mutation in a HEXB gene resulting in reduced or loss of function of a hexosaminidase beta-subunit of the subject. 
     
     
         33 . The method of any one of  claims 29 to 32 , wherein the rAAV is administered by intracranial injection, intracerebral injection, or injection into the CSF via the cerebral ventricular system, cisterna magna, or intrathecal space. 
     
     
         34 . The method of any one of  claims 29 to 33 , wherein the subject is administered the isolated nucleic acid or the rAAV during a pre-symptomatic stage of the lysosomal storage disease. 
     
     
         35 . The method of  claim 34 , wherein the pre-symptomatic stage of the lysosomal storage disease occurs between birth (e.g., perinatal) and 4-weeks of age.